U.S. patent application number 11/633129 was filed with the patent office on 2007-04-19 for enhanced circulation effector composition and method.
This patent application is currently assigned to Alza Corporation. Invention is credited to Yechezkel Barenholz, Herve Bercovier, Francis J. Martin, Martin C. Woodle, Samuel Zalipsky.
Application Number | 20070087047 11/633129 |
Document ID | / |
Family ID | 27364849 |
Filed Date | 2007-04-19 |
United States Patent
Application |
20070087047 |
Kind Code |
A1 |
Zalipsky; Samuel ; et
al. |
April 19, 2007 |
Enhanced circulation effector composition and method
Abstract
A liposome composition comprising small, surface-bound effector
molecules is disclosed. The liposomes have a surface layer of
hydrophilic polymer chains, for enhanced circulation time in the
bloodstream. The effector molecules are attached to the distal ends
of the polymer chains. In one embodiment, the effector is polymyxin
B, for treatment of septic shock.
Inventors: |
Zalipsky; Samuel; (Redwood
City, CA) ; Woodle; Martin C.; (Menlo Park, CA)
; Martin; Francis J.; (San Francisco, CA) ;
Barenholz; Yechezkel; (Jerusalem, IL) ; Bercovier;
Herve; (Jerusalem, IL) |
Correspondence
Address: |
PERKINS COIE LLP
P.O. BOX 2168
MENLO PARK
CA
94026
US
|
Assignee: |
Alza Corporation
|
Family ID: |
27364849 |
Appl. No.: |
11/633129 |
Filed: |
December 1, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10832636 |
Apr 26, 2004 |
7150882 |
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11633129 |
Dec 1, 2006 |
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10438502 |
May 14, 2003 |
7160554 |
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10832636 |
Apr 26, 2004 |
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09877978 |
Jun 8, 2001 |
6586002 |
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10438502 |
May 14, 2003 |
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08480332 |
Jun 7, 1995 |
6180134 |
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09877978 |
Jun 8, 2001 |
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08316436 |
Sep 29, 1994 |
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08480332 |
Jun 7, 1995 |
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08035443 |
Mar 23, 1993 |
6326353 |
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08316436 |
Sep 29, 1994 |
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Current U.S.
Class: |
424/450 ;
424/85.2; 424/85.5; 424/85.6; 424/85.7 |
Current CPC
Class: |
A61K 38/1774 20130101;
A61K 47/62 20170801; A61K 38/12 20130101; A61K 47/6911 20170801;
A61K 47/61 20170801; A61K 9/1271 20130101; A61K 31/00 20130101;
Y10S 424/812 20130101; A61K 9/0019 20130101; A61K 38/12 20130101;
A61K 2300/00 20130101; A61K 38/1774 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/450 ;
424/085.2; 424/085.5; 424/085.6; 424/085.7 |
International
Class: |
A61K 38/21 20060101
A61K038/21; A61K 38/20 20060101 A61K038/20; A61K 9/127 20060101
A61K009/127 |
Claims
1. A liposome composition, comprising liposomes, each having an
outer layer of a hydrophilic, and an effector molecule attached to
the distal ends of said chains, said effector molecule having
binding affinity to a cell receptor, wherein said liposome-bound
effector molecule binds to the cell receptor and sterically hinders
the cell receptor.
2. The composition of claim 1 wherein the effector molecule is
selected from the group consisting of F.sub.ab antibody fragments,
cytokines, cellular growth factors, peptide hormones,
monosaccharides, polysaccharides, IL-1 inhibitors, ELAM-1 binding
inhibitors, and limulus antilipopolysaccharide factor (LALF).
3. The composition of claim 2 wherein the polysaccharide is sialyl
Lewis.sup.x.
4. The composition of claim 2 wherein the cytokine is selected from
the group consisting of interferons, interleukins, TNF,
transforming growth factor .beta., lymphotoxin, GM-CSF, and
G-CSF.
5. The composition of claim 4 wherein the interferon is selected
from the group consisting of IFN-alpha, IFN-beta, and
IFN-gamma.
6. The composition of claim 4 wherein the interleukin is selected
from the group consisting of IL-1.alpha., IL-1.beta., IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, and IL-8.
7. A liposome composition for use in treating a condition mediated
by binding of one binding member to a second binding member,
comprising liposomes, each having an outer layer of a hydrophilic
polymer, an effector molecule attached to the distal ends of said
chains, said effector molecule having binding affinity to a cell
receptor, wherein said liposome-bound effector molecule binds to
the cell receptor and sterically hinders the cell receptor.
8. The composition of claim 7 wherein the effector molecule is
selected from the group consisting of F.sub.ab antibody fragments,
cytokines, cellular growth factors, peptide hormones,
monosaccharides, polysaccharides, IL-1 inhibitors, ELAM-1 binding
inhibitors, and limulus antilipopolysaccharide factor (LALF).
9. The composition of claim 8 wherein the polysaccharide is sialyl
Lewis.sup.x.
10. The composition of claim 8 wherein the cytokine is selected
from the group consisting of interferons, interleukins, TNF,
transforming growth factor .beta., lymphotoxin, GM-CSF, and
G-CSF.
11. The composition of claim 10 wherein the interferon is selected
from the group consisting of IFN-alpha, IFN-beta, and
IFN-gamma.
12. The composition of claim 10 wherein the interleukin is selected
from the group consisting of IL-1.alpha., IL-1.beta., IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, and IL-8.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 10/832,636 filed Apr. 26, 2004, now allowed; which is a
continuation of U.S. application Ser. No. 10/438,502 filed May 14,
2003, now pending; which is a continuation of U.S. application Ser.
No. 09/877,978 filed Jun. 8, 2001, now U.S. Pat. No. 6,586,002;
which is a continuation of U.S. application Ser. No. 08/480,332
filed Jun. 7, 1995, now U.S. Pat. No. 6,180,134; which is a
continuation-in-part of U.S. application Ser. No. 08/316,436 filed
Sep. 29, 1994, now abandoned; which is a continuation-in-part of
U.S. application Ser. No. 08/035,443 filed Mar. 23, 1993, now U.S.
Pat. No. 6,326,353; all of which are incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to an enhanced-circulation
effector composition and method for treating a subject with small
effector molecules which are normally subject to rapid renal
clearance from the bloodstream.
BACKGROUND OF THE INVENTION
[0003] A number of emerging or current therapies involve
intravenous injection of small (less than 50 Kdaltons) protein,
polypeptide or polysaccharide effectors. Such effectors can include
F.sub.ab antibody fragments for use in active immunity, cytokines
and cellular growth factors for stimulating immunological
inflammatory responses, hormones, and polysaccharides, which are
capable of interacting with endothelial cell receptors to
competitively block neutrophil binding to activated endothelial
cells lining the blood vessel (Katre, N. V., et al., Proc. Natl.
Acad. Sci. USA 84:1487-1491 (1987); Philips, M. L., et al., Science
250:1130-1132 (1990); Waldmann, T. A., Annu. Rev. Immunol.
10:675-704 (1992)).
[0004] Other small polypeptide effectors have been proposed for use
in blocking viral infection of target cells in the blood, such as a
CD4+ glycopeptide which is effective to inhibit binding of human
immunodeficiency virus (HIV) to CD4.sup.+ cells (Capon, D. J. and
Ward, R. H. R., Ann. Rev. Immunol. 9:649-678 (1991); Janeway, C.
A., Ann. Rev. Immunol. 10:645-674 (1992)).
[0005] Polymyxin B, a small basic peptide which is rapidly excreted
by the kidneys, is known to react with and neutralize gram-negative
bacterial endotoxins, specifically E. coli 0111:B4 liposaccharide
(LPS) (Baldwin, G., et al., J. Infect. Diseas. 164:542-549 (1991)).
It is not often administered parenterally as a treatment for septic
shock syndrome, because high doses of polymyxin B are required for
effective treatment. High doses can be fatal, due to renal
toxicity, making advanced stages of septic shock difficult to
treat.
[0006] The problem of rapid renal clearance observed with polymyxin
B is also applicable to other small peptides, such as those
discussed above, which have been used for parenteral treatment of
disease. In general, circulating proteins which are smaller than
about 50-60 Kdaltons will be cleared by the kidneys with a lifetime
of less than 1-2 hours.
[0007] In some cases, peptide molecular weight can be increased
above the threshold 50-60 Kdalton size by derivatizing the peptide
with biologically compatible polymers, such as polyethyleneglycol
(PEG) (e.g., U.S. Pat. No. 4,179,337). However, this strategy may
not always be effective for small effectors, e.g., those with
molecular weights less than about 5-10 Kdalton. Moreover,
derivatizing a polypeptide with a plurality of PEG chains may
destroy or reduce the polypeptide activity, and/or mask key
activity sites of the polypeptide.
SUMMARY OF THE INVENTION
[0008] The invention includes, in one aspect, a liposome
composition for use in treating a subject with a polypeptide or
polysaccharide effector which is effective as a pharmacological
agent when circulating in free form in the bloodstream, but which
is rapidly removed from the bloodstream by renal clearance in free
form. The composition includes liposomes having an outer surface
layer of polyethylene glycol chains and the effector covalently
attached to the distal ends of the chains. A preferred polymer is
polyethylene glycol having a molecular weight between about 1,000
and 10,000 daltons.
[0009] Preferred effectors include:
[0010] (a) an antibody F.sub.ab fragment having neutralizing
activity against a given pathogen present in the bloodstream, for
use in treating the subject for infection by the pathogen;
[0011] (b) a CD4 glycoprotein, for use in treating the subject for
infection by human immunodeficiency virus (HIV);
[0012] (c) a cytokine or a cellular growth factor, for use in
stimulating an immune response in the subject;
[0013] (d) a polysaccharide which binds to endothelial leukocyte
adhesion molecule (ELAM), for use in treating inflammation related
to neutrophil recruitment and tissue infiltration;
[0014] (e) IL-1 inhibitor or IL-1RA, for treating a subject to
achieve immune-response suppression;
[0015] (f) polymyxin B or polymyxin B decapeptide, for treating the
subject for septic shock;
[0016] (g) a peptide hormone, for treating a subject to regulate
cellular growth; and
[0017] (h) a peptide, for inhibiting a ligand-receptor cell-binding
event.
[0018] In one specific embodiment, the invention includes a method
of preventing progression of gram-negative bacteremia to septic
shock and a method of treating acute septic shock by administering
to a subject, a liposome composition containing liposomes having an
outer layer of polyethylene glycol (PEG) chains and polymyxin B
attached to the distal ends of the polymer chains.
[0019] In another aspect, the invention includes a liposome
composition for use in preventing rapid removal from the
bloodstream of a polypeptide or polysaccharide effector by renal
clearance. The composition includes liposomes having an outer layer
of polyethylene glycol chains, and attached to the distal ends of
the chains, is one of the above effectors (a)-(h). These and other
objects and features of the invention will become more fully
apparent when the following detailed description of the invention
is read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1A-1B show steps for the synthesis of a maleimide of a
DSPE carbamide of polyethylene glycol (PEG) bis(amine);
[0021] FIG. 2 shows steps for the synthesis of a disulfide
linkage-containing propionamide of a DSPE (distearyl
phosphatidylethanolamine) carbamide of polyethylene glycol (PEG)
bis(amine);
[0022] FIG. 3 shows a synthetic scheme for the preparation of a
PEG-derivatized PE (phosphatidylethanolamine) containing a terminal
aldehyde group;
[0023] FIG. 4 illustrates a synthetic scheme for forming a
PEG-derivatized DSPE having a reactive maleimide group at the PEG
terminus;
[0024] FIG. 5 illustrates an exemplary method for forming a
PEG-derivatized DSPE containing a bromoacetamide group at the
polymer end;
[0025] FIG. 6 shows an exemplary method for synthesizing a
derivatized DSPE lipid with a PEG chain functionalized to contain a
terminal hydrazide group;
[0026] FIGS. 7A-7D show steps in the synthesis of a PEG-derivatized
DSPE lipid containing a reactive group at the polymer end (FIG. 7A)
which can be used to couple to a variety of amine containing groups
(7B-7D);
[0027] FIG. 8 shows one synthetic approach for forming DSPE
derivatized by a PEG spacer chain having a terminal hydrazide group
(shown in protected form);
[0028] FIG. 9 shows the covalent coupling of a
sulfhydryl-containing peptide to the terminal maleimide group of a
DSPE carbamide PEG derivative;
[0029] FIG. 10 shows the covalent coupling of a
sulfhydryl-containing peptide via formation of a disulfide bond to
a DSPE carbamide of a terminally functionalized PEG containing a
reactive disulfide linkage derived from SPDP (N-succinimidyl
3-(2-pyridyldithio)-propionate);
[0030] FIG. 11 shows the covalent coupling of a peptide through the
aldehyde group of an ethylene-linked derivative of DSPE carbamide
of PEG by reductive amination;
[0031] FIG. 12 shows a plot of a time course of gallium-67 labelled
liposomes composed of hydrazide PEG-DSPE, partially hydrogenated
egg phosphatidylcholine (PHEPC), and cholesterol (PEG-HZ fluid
liposomes) or hydrazide PEG-DSPE, hydrogenated serum
phosphatidylcholine (HSPC), and cholesterol (PEG-HZ rigid
liposomes) in the bloodstream; and
[0032] FIG. 13 shows the amino acid sequences for peptides
identified by SEQ ID NOS:1-10, in conventional single-letter amino
acid code.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0033] Unless otherwise indicated, the terms below have the
following meaning:
[0034] "Vesicle-forming lipid" refers to any lipid capable of
forming part of a stable micelle or liposome composition and
typically including one or two hydrophobic acyl hydrocarbon chains
or a steroid group and may contain a chemically reactive group,
such as an amine, acid, ester, aldehyde or alcohol, at its polar
head group.
[0035] "Effector" refers to polypeptides, mono or polysaccharides,
and glycopeptides. Polypeptides, polysaccharides or glycopeptides
may have sizes up to about 50-60 Kdaltons.
II. Effector Composition
[0036] The invention includes, in one aspect, a liposome
composition for use in treating a subject with a small polypeptide
or polysaccharide effector molecule which is effective as a
pharmacological agent when circulating in free form in the
bloodstream, but which is rapidly removed from the bloodstream by
renal clearance. The composition includes a liposomal carrier
composed of liposomes having an outer surface layer formed of
hydrophilic polymer chains, e.g., PEG. The effector is attached to
the distal ends of the polymer chains in at least a portion of the
derivatized vesicle-forming lipid. The effector is attached to the
distal end of a polymer chain to preserve the biological activity
of the effector, such as behaving as a member of a ligand-receptor
binding pair. The preparation of the composition follows the
general procedures below.
[0037] A. Lipid Components
[0038] The liposomal carrier of the composition is composed of
three general types of vesicle-forming lipid components. The first
includes vesicle-forming lipids which will form the bulk of the
vesicle structure in the liposome.
[0039] Generally, these vesicle-forming lipids include any
amphipathic lipids having hydrophobic and polar head group
moieties. Such a vesicle-forming lipid for use in the present
invention is one which (a) can form spontaneously into bilayer
vesicles in water, as exemplified by the phospholipids, or (b) is
stably incorporated into lipid bilayers, with its hydrophobic
moiety in contact with the interior, hydrophobic region of the
bilayer membrane, and its polar head group moiety oriented toward
the exterior, polar surface of the membrane.
[0040] The vesicle-forming lipids of this type are preferably ones
having two hydrocarbon chains, typically acyl chains, and a polar
head group. Included in this class are the phospholipids, such as
phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidic acid (PA), phosphatidylinositol (PI), and
sphingomyelin (SM), where the two hydrocarbon chains are typically
between about 14-22 carbon atoms in length, and have varying
degrees of unsaturation. The above-described lipids and
phospholipids whose acyl chains have varying degrees of saturation
can be obtained commercially or prepared according to published
methods. Other suitable lipids include glycolipids and sterols such
as cholesterol.
[0041] The second general component includes a vesicle-forming
lipid which is derivatized with a polymer chain. Vesicle-forming
lipids for use as the second general vesicle-forming lipid
component (e.g., are suitable for derivatization with a polymer)
are any of those described for the first general vesicle-forming
lipid component. Vesicle forming lipids with diacyl chains, such as
phospholipids, are preferred. One exemplary phospholipid is
phosphatidylethanolamine (PE), which provides a reactive amino
group which is convenient for coupling to the activated polymers.
An exemplary PE is distearyl PE (DSPE).
[0042] A preferred polymer for use in forming the derivatized lipid
component is polyethyleneglycol (PEG), preferably a PEG chain
having a molecular weight between 1,000-10,000 daltons, more
preferably between 2,000 and 5,000 daltons. Other hydrophilic
polymers which may be suitable include polyvinylpyrrolidone,
polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl
methacrylamide, polymethacrylamide and polydimethylacrylamide,
polylactic acid, polyglycolic acid, and derivatized celluloses,
such as hydroxymethylcellulose or hydroxyethylcellulose.
[0043] Additionally, block copolymers or random copolymers of these
polymers, particularly including PEG segments, may be suitable.
Methods for preparing lipids derivatized with hydrophilic polymers,
such as PEG, are well known e.g., as described in co-owned U.S.
Pat. No. 5,013,556.
[0044] The third general vesicle-forming lipid component is a lipid
anchor by which the effector is anchored to the liposomes, through
a polymer chain in the anchor. Additionally, the effector is
positioned at the distal end of the polymer chain in such a way so
that the biological activity of the effector is not lost. The lipid
anchor has a hydrophobic moiety which serves to anchor the lipid in
the outer layer of the liposome bilayer surface, a polar head group
to which the interior end of the polymer is covalently attached,
and a free (exterior) polymer end which is or can be activated for
covalent coupling to the effector. Methods for preparing lipid
anchor molecules of this types are described below.
[0045] B. Liposome Preparation
[0046] The liposomes may be prepared by a variety of techniques,
such as those detailed in Szoka, F., Jr., et al., Ann. Rev.
Biophys. Bioeng. 9:467 (1980). Multilamellar vesicles (MLVs) can be
formed by simple lipid-film hydration techniques. In this
procedure, a mixture of liposome-forming lipids of the type
detailed above dissolved in a suitable organic solvent is
evaporated in a vessel to form a thin film, which is then covered
by an aqueous medium. The lipid film hydrates to form MLVs,
typically with sizes between about 0.1 to 10 microns.
[0047] The lipid components used in forming the liposomes are
preferably present in a molar ratio of about 70-90 percent vesicle
forming lipids, 1-25 percent polymer derivatized lipid, and 0.1-5
percent lipid anchor. One exemplary formulation includes 50-70 mole
percent underivatized PE, 20-40 mole percent cholesterol, 0.1-1
mole percent of a PE-PEG (3500) polymer with a chemically reactive
group at its free end for effector coupling, 5-10 mole percent PE
derivatized with PEG 3500 polymer chains, and 1 mole percent
.alpha.-tocopherol.
[0048] The liposomes are preferably prepared to have substantially
homogeneous sizes in a selected size range, typically between about
0.03 to 0.5 microns. One effective sizing method for REVs and MLVs
involves extruding an aqueous suspension of the liposomes through a
series of polycarbonate membranes having a selected uniform pore
size in the range of 0.03 to 0.2 micron, typically 0.05, 0.08, 0.1,
or 0.2 microns. The pore size of the membrane corresponds roughly
to the largest sizes of liposomes produced by extrusion through
that membrane, particularly where the preparation is extruded two
or more times through the same membrane. Homogenization methods are
also useful for down-sizing liposomes to sizes of 100 nm or less
(Martin, F. J., in SPECIALIZED DRUG DELIVERY SYSTEMS-MANUFACTURING
AND PRODUCTION TECHNOLOGY, (P. Tyle, Ed.) Marcel Dekker, New York,
pp. 267-316 (1990)).
[0049] C. Effector Component
[0050] The effector in the composition is a therapeutic
polypeptide, mono or polysaccharide, or glycopeptide characterized,
when administered intravenously in free form, by rapid clearance
from the bloodstream, typically within 1-2 hours. The effector
itself is effective as a pharmacological agent when circulating in
free form in the bloodstream. Below are described preferred
effectors for use in the invention.
[0051] 1. F.sub.ab Fragment. The F.sub.ab fragment is one which has
neutralizing activity against a given pathogen. The composition is
used as a passive vaccine effective to provide humoral immunity
against one of a variety of selected pathogenic antigens.
[0052] F.sub.ab fragments of neutralizing antibodies can be
prepared according to conventional methods (Harlow, E., et al., in
ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Press,
Plainville, N.Y., (1988)). The fragment is preferably from a
humanized monoclonal antibody (M.sub.ab). Such antibodies can be
prepared by published recombinant DNA methods (Larrick, J. W., et
al., Methods in Immunology 2:106 (1991)). The antibody is
preferably coupled to liposomal hydrophilic polymer groups via
sulfhydryl linkages, as described above.
[0053] 2. CD4 Glycoprotein Effector. The CD4 glycopeptide is a
region of the CD4 receptor of CD4+ T cells (Capon and Ward). The
effector acts to block HIV infection of CD4+ T cells by blocking
gp120-mediated HIV binding to the CD4 receptor. The effector can be
produced according to known recombinant methods (Maniatis, T., et
al., in MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989)).
[0054] 3. Cytokines. The cytokines given in Table 1 below are
examples of cytokines which are useful in the present invention.
The cytokines may be obtained by recombinant production methods,
according to published procedures. The therapeutic uses of the
individual cytokines have been described in the literature (see,
for example, Abbas, A. K., et al., in CELLULAR AND MOLECULAR
IMMUNOLOGY, W. B. Saunders Company Harcourt Brace Jovanovich,
Philadelphia (1991)). Some cytokine effectors may be administered
on a short term basis to enhance a weak immunogenic or weak
microbicidal response. The effectors may be administered on a long
term basis as part of a therapy treatment for cancer or AIDS
(Waldmann). TABLE-US-00001 TABLE 1 CYTOKINE POLYPEPTIDE SIZE A.
Mediators of Natural Immunity IFN-alpha 18 kD (monomer) IFN-beta 20
kD (monomer) Tumor necrosis factor (TNF) 17 kD (homotrimer)
Interleukin-1 (alpha and beta) 17 kD (monomer) Interleukin-6 26 kD
(monomer) Interleukins-8's 8-10 (monomer or dimer) B. Mediators of
Lymphocyte Activation, Growth and Differentiation Interleukin-2
14-17 kD (monomer) Interleukin-4 20 kD (monomer) Transforming
growth factor (beta) 14 kD (monomer or dimer) C. Mediators of
Effector Cell Adhesion Gammma Interferon 21-24 kD (homodimer)
Lymphotoxin 24 kD (homotrimer) Interleukin-5 20 kD (monomer) D.
Mediators of Immature Leukocyte Growth and Differentiation
Interleukin-3 20-26 kD (monomer) Granulocyte-macrophage Colony 22
kD (monomer) Stimulating Factor Macrophage Colony Stimulating 40 kD
(dimer) Factor Granulocyte CSF 19 kD (monomer) Interleukin-7 25 kD
(monomer)
[0055] 4. ELAM-1 Binding Inhibitor. Inflammation causes the
expression of a polypeptide, endothelial leukocyte adhesion
molecule-1 (ELAM-1), on the surface of endothelial cells of blood
vessels, adjacent to sites of inflammation. ELAM-1, in turn,
recognizes and binds a polysaccharide moiety, sialyl Lewis.sup.x,
on surfaces of neutrophils and recruits neutrophils to sites of
inflammation. By preventing the recognition and binding of
neutrophils by ELAM-1, excessive inflammatory responses due to
conditions such as reperfusion injury, septic shock, and chronic
inflammatory diseases, can be avoided.
[0056] In this embodiment, the effector is the tetrasaccharide,
sialyl Lewis.sup.x, recognized by ELAM-1 (Phillips, M. L., et al.,
Science 250:1130-1132 (1990)), for therapeutical use in preventing
excessive recruitment of neutrophils to sites of inflammation in
the blood stream. The effector is produced by the glycosylation
mutants of Chinese hamster ovary (CHO) cells, and may be obtained
in purified form from the cultured cells (Phillips). Alternatively,
the effector is produced by chemical and/or enzymatic synthesis
(Borman, S., Chem. Eng. News, December 7:25-28 (1992); Ichikawa, Y.
et al., J. Am. Chem. Soc. 114:9283-9298 (1992)).
[0057] 5. Inhibitors of IL-1 Activity. The effector in this
embodiment is an IL-1 inhibitor, or IL-1 receptor antagonist
(IL1PA), which blocks binding of IL-1 to receptors on lymphocyte
cell surfaces (Stylianou, E., et al., J. Biol. Chem.
267:15836-15841 (1992)).
[0058] IL-1 production is stimulated by both endotoxins which cause
septic shock and exotoxins which cause toxic shock syndrome
(Dinarello, C. A., Blood 77(8):1627-1650 (1991)). IL-1 production
during septic shock or toxic shock may exacerbate the clinical
symptoms observed in patients. Therefore, use of an IL-1 inhibitor
effector to decrease the clinical symptoms associated with either
toxic shock or septic shock may be beneficial.
[0059] IL-1 inhibitor is a 52 to 66 Kd polypeptide that binds
specifically to IL-1 to inhibit its immunostimulatory responses.
IL1RA is a 23 to 25 KD polypeptide that competes with binding of
IL-1 to its cell surface receptors to inhibit IL-1's
immunostimulatory responses.
[0060] 6. Polymyxin B. This effector is a cationic detergent with a
hydrophobic portion (6-methyloctanoyl) and a short basic
decapeptide portion. Polymyxin B reacts with and neutralizes
gram-negative bacterial endotoxins, specifically E. coli 0111:B4
liposaccharide (LPS) (Baldwin, et al.). Polymyxin B is used in the
treatment of gram-negative bacterial infections. Since polymyxin B
must be administered frequently and in high doses because of its
rapid clearance from the bloodstream, it causes severe irreversible
kidney damage. Polymyxin B can be chemically synthesized or
isolated from spore-forming gram-positive bacilli, such as Bacillus
polymyxa.
[0061] Alternatively, the effector is an 11.8 Kdalton peptide
isolated from amebocytes of Limulus polyphemus, limulus
antilipopolysaccharide factor (LALF). LALF neutralizes
meningococcal lipooligosaccharide, as well as other gram-negative
endotoxins, and can be used to treat gram-negative sepsis
(Wainright, N. R., et al., In CELLULAR AND MOLECULAR ASPECTS OF
ENDOTOXIN REACTIONS (Nowotny, A., et al., Eds.) Elsevier Science
Publishers B. V., p. 315 (1990)).
[0062] 7. Peptide Hormone. This effector can be used in the
treatment of various diseases. In one embodiment, the effector is
parathyroid hormone (PTH) which is 84 amino acids in length and can
inhibit osteoblast division. Certain bone cancers are characterized
by uncontrolled osteoblast division (Kano, J., et al., Biochem.
Biophys. Res. Comm. 179:97-101 (1991)). Alternatively, the peptide
hormone can be used to target a liposome to cells that contain
receptors for a specific peptide hormone.
[0063] D. Attachment of Effector to Liposome Carrier
[0064] For effector attachment to liposome carriers, the free
polymer end of a lipid anchor is activated prior to effector
coupling. In the following specific examples, both lipid anchor
formation and activation reactions are described. The reactions are
shown with respect to the free lipid, either
distearylphosphatidyl-ethanolamine (DSPE) or PE.
[0065] The activated lipid anchors are then incorporated into
liposomal carriers, as described above.
[0066] One advantage of activating the PEG terminal group of the
lipid anchor prior to liposome formation is that a broader range of
reaction solvents and reaction conditions may be employed. Further,
the liposomes themselves are not exposed to the activating
reagents. Thus, the need to remove reagent contaminants from the
liposomes is avoided.
[0067] It will also be appreciated that the activation reactions
may be performed after lipid anchor incorporation into liposomal
carriers. In some coupling reactions it may be more desirable to
activate the terminal PEG groups on preformed liposomes. One
advantage of this approach is that the activation reaction is
confined to the outer, surface-accessible lipids, and thus the
activated groups can be completely quenched prior to use of the
composition in therapy. The approach is also preferred for
reactions in which the activated PEG termini are unstable in
water.
[0068] FIGS. 1A-1B show the synthesis of DSPE derivatized with a
PEG chain and having an activated maleimide group at the chain's
free end. Initially, PEG bis(amine) (compound I) is reacted with
2-nitrobenzene sulfonyl chloride to generate the monoprotected
product (compound II). Compound II is reacted with carbonyl
diimidazole in triethylamine (TEA) to form the imidazole carbamide
(e.g., urea) of the mono 2-nitrobenzenesulfonamide (compound
III).
[0069] Compound III is reacted with DSPE in TEA to form the
derivatized PE lipid protected at one end with 2-nitrobenzyl
sulfonyl chloride. The protecting group is removed by treatment
with acid to give the DSPE-PEG product (compound IV) having a
terminal amine on the PEG chain. Reaction with maleic anhydride
gives the corresponding end-functionalized product (compound V),
which on reaction with acetic anhydride gives the desired
DSPE-PEG-maleimide product (compound VI). Details of the reactions
are given in Example 1.
[0070] The compound is reactive with sulfhydryl groups, for
coupling polypeptides through a thioether linkage, as illustrated
in FIG. 9.
[0071] FIG. 2 illustrates an exemplary synthesis of another
derivatized lipid useful for coupling to sulfhydryl-containing
polypeptides. Here the DSPE-PEG lipid (compound IV) described above
is treated with N-succinimidyl-3-(2-pyridyldithio)propionamide,
SPDP, (compound VII) to form the anchor DSPE-PEG lipid (compound
VIII). The compound can, for example, react with a sulfhydryl group
of a peptide to thereby couple the peptide to the lipid through a
disulfide linkage as illustrated in FIG. 10.
[0072] Another synthetic approach for coupling a protected
polyalkylether to a lipid amine is shown in FIG. 3. In this
reaction scheme, PEG (compound IX) is initially protected at one of
its terminal OH ends by a trimethylsilyl group, as shown at the top
of FIG. 3. The monoprotected PEG (compound X) is reacted with the
anhydride of trifluoromethyl sulfonate to activate the free PEG end
with trifluoromethyl sulfonate (compound XI). Reaction of the
activated PEG compound with a lipid amine, such as PE, in the
presence of triethylamine, and release of the trimethylsilyl
protecting group by acid treatment, gives the PE-PEG derivative
(compound XII). This compound contains a terminal alcohol group
which is then oxidized in the presence of dimethylsulfoxide (DMSO)
and acetic anhydride to form an aldehyde group (compound XIII)
which can be coupled to a peptide via reductive amination, as
illustrated in FIG. 11. Reaction details are given in Example
2.
[0073] More generally, the derivatized lipid components can be
prepared to include a lipid-polymer linkage, such as a peptide,
ester, or disulfide linkage, which can be cleaved under selective
physiological conditions, such as in the presence of peptidase or
esterase enzymes or reducing agents, such as glutathione, present
intracellularly.
[0074] An alternative general method for preparing lipid
derivatives of PEG suitable for coupling to effector molecules
involves using .alpha.-amino-.omega.-carboxy derivatives of PEG
(such as compound XIV) as starting materials. This alternative
approach is illustrated in FIGS. 4, 5, and 6.
[0075] Methods for preparing heterobifunctional PEG derivatives
such as compound XIV have been described by Zalipsky, S., et al.,
Polymer Preprints 27(1):1 (1986); Zalipsky, S., et al., J.
Bioactive Compat. Polym. 5:227 (1990)). In the reaction scheme
shown in FIG. 4, an .alpha.-amino-.omega.-carboxy functionalized
PEG (Zalipsky, et al., 1986) is reacted with
N-(.gamma.-maleimidobutyryl-oxy)succinimide ester (GMBS, Pierce),
using an excess of GMBS. The terminal carboxyl group of the
resulting maleimido-PEG (compound XV) is then reacted with a lipid
amine, such as PE or DSPE, in the presence of N-hydroxysuccinimide,
to link the PEG to the lipid through an amide linkage (compound
XVI). The maleimido group at the "free" end of the polymer is
reactive towards thiol-containing ligands, proteins, e.g.,
immunoglobulins and fragments thereof.
[0076] A related scheme is illustrated in FIG. 5, which shows
introduction of a terminal bromoacetamide group in an
.alpha.-amino-.omega.-carboxy-functionalized PEG. In the approach
shown, a derivative of PEG is reacted with bromoacetyl
N-hydroxysuccinimide ester. The bromoacetamido-functionalized PEG
is then coupled to a suitable lipid amine, such as PE or DSPE, as
above, to form the derivatized lipid (compound XVIII). The
bromoacetamide group, being more selective and more stable than a
maleimide group, allows greater flexibility in the methods used for
liposome formation and loading.
[0077] The reaction scheme shown in FIG. 6 illustrates the
preparation of a derivatized lipid in which the free PEG end is
functionalized to contain a hydrazide. In the reaction illustrated
in FIG. 6, an .alpha.-hydroxy-.omega.-carboxylic acid PEG
derivative (compound XIX) (Zalipsky, et al., 1990) is esterified
with methanol to protect the terminal acid group by formation of
the corresponding ester (compound XX). The terminal hydroxyl group
is then converted into a functional group reactive towards primary
amines (Zalipsky, S., et al., in POLY (ETHYLENE GLYCOL) CHEMISTRY:
BIOTECHNICAL AND BIOMEDICAL APPLICATIONS (J. M. Harris, Ed.) Plenum
Press, pg. 347-370 (1992a)), for example, a succinimidyl carbonate
(SC) derivative (compound XXI). This compound is formed, for
example, by reacting compound XX with phosgene followed by
subsequent reaction with N-hydroxysuccinimide (Zalipsky, S., et
al., Biotechnol. Appi. Biochem. 15:100 (1992b)). The resulting
activated PEG compound, SC-PEG-C(O)NHCH.sub.2CO.sub.2-Me (compound
XXI) reacts with a lipid amine such as PE or DSPE at the reactive
succinimidyl carbonate group to form the functionalized lipid,
DSPE-PEG-C(O)NHCH.sub.2CO.sub.2-Me (compound XXII). The methyl
ester can be readily hydrazinolyzed to yield
DSPE-NHCO.sub.2-PEG-C(O)NHCH.sub.2C(O)--N.sub.2H.sub.3 (compound
XXIII), as shown. This hydrazide-containing PEG-lipid is
incorporated into liposomes by conventional methods. The hydrazide
group can be used for attachment of aldehyde or ketone containing
effector molecules.
[0078] Such carbonyl groups exist or can be easily generated on
numerous carbohydrate containing molecules, e.g. oligosaccharides,
nucleotides, low molecular weight glycosides, lectins,
immunoglobulins and other glycoproteins by chemical (periodate
oxidation) or enzymatic reactions (galactose oxidase). The linkages
formed, hydrazones, are reasonably stable at pH.gtoreq.7.5, but are
cleavable by acid catalyzed hydrolysis at lower pH values. These
linkages can be stabilized by reduction, e.g., with sodium
cyanoborohydride. An advantage of this approach is the stability of
hydrazide groups which allows the use of a wide array of liposome
formulations and loading protocols.
[0079] Alternatively, as illustrated in FIG. 7A, an
.alpha.-hydroxy-.omega.-carboxy derivative of PEG (compound XIX)
can be coupled to a lipid containing a terminal amino group, e.g.,
DSPE, by reaction with N-hydroxysuccinimide in the presence of a
coupling agent such as dicyclohexylcarbodiimide, DCC. The resulting
intermediate, the N-hydroxysuccinimide (NHS) ester of
.alpha.-hydroxy-PEG, is then suitable for coupling to an amino-end
containing lipid such as DSPE by displacement of the NHS group to
form a .alpha.-hydroxy-PEG-DSPE conjugate, linked by an amide bond
(compound XXIV, FIG. 7A). The .alpha.-hydroxy group of PEG can then
be further activated, such as by reaction with disuccinimidyl
carbonate (DSC), to form an .alpha.-succinimidyl carbonate-PEG-DSPE
compound (compound XXV) suitable for coupling to a variety of
compounds containing reactive amino groups.
[0080] Preparation of compound XXIV is described in Example 4.
Amino-group containing compounds for coupling to such
functionalized lipids will also possess at least one other
functional group to which effector molecules may be attached. The
attachment of the effector molecules may occur before or after
liposome formation.
[0081] In one case, as illustrated in FIG. 7B, the SC-PEG-DSPE is
reacted with 2-aminoethanedithiopyridine. The derivative formed
(compound XXVI) can be used in the following manner. The
dithiopyridine group is reactive towards thiol-containing molecules
but is also quite stable under a variety of conditions. Using mild
reducing agents, e.g., .beta.-mercaptoethanol, it is possible to
convert the dithiopyridine groups on the liposomes into free
thiols, which in turn can be used in various conjugation procedures
involving ligands containing reactive maleimido or bromoacetate
groups or reactive mixed disulfide groups such as
dithiopyridine.
[0082] In the reaction illustrated in FIG. 7C, the SC-PEG-DSPE is
reacted with 3-amino-1,2-propanediol, producing a diol terminated
PEG-lipid (compound XXVII). After incorporation into a liposome,
the diol can be oxidized (e.g., with periodate) under mild
conditions ([I0.sub.4-].ltoreq.10 mM, 4.degree. C.) to provide a
reactive aldehyde. The aldehyde containing PEG-liposomes will react
irreversibly with a variety of amino-containing effector molecules
in the presence of a reducing agent such as sodium
cyanoborohydride.
[0083] In the reaction illustrated in FIG. 7D, SC-PEG-DSPE is
coupled to a galactosamine. The galactose residue on the
derivatized lipid (compound XXVIII) can then be enzymatically
oxidized by galactose oxidase. The aldehyde bearing PEG-liposomes
obtained by this process can be used for conjugation with
amino-group containing effector molecules. In addition to the
mildness of the reaction conditions, the aldehyde groups are
generated solely on the outer surface of the liposome.
[0084] Additionally, there is evidence that oxidized galactose
residues are useful for stimulation of the immune system,
specifically for T cell activation. A liposome having oxidized
galactose residues on its surface is likely to act as an adjuvant
and might be useful in vaccines (Zheng, B., et al., Science
256:1560-1563 (1992)).
[0085] In another procedure, illustrated in FIG. 8 and described in
Example 5, DSPE-PEG-hydrazide is prepared. First PEG is reacted
with ethyl isocyanatoacetate in the presence of triethylamine to
generate mono and di-end carboxylated species of PEG, where the
carboxylic acid functions are connected to the PEG skeleton via
intervening carbamate bonds. The monocarboxylated species is
purified by ion-exchange chromatography on DEAE-Sephadex (compound
XXIX, identical to compound XIX). Compound XXIX is reacted with
tert-butyl carbazate to generate the
.omega.-hydroxy-.alpha.-Boc-hydrazide derivative of PEG (compound
XXX). The hydroxyl terminus of PEG is then activated by reaction
with disuccinimidyl carbonate to form compound XXXI prior to
reaction with DSPE to generate the desired lipid-PEG-.alpha.-Boc
hydrazide product (compound XXXII). Compound XXXII is deprotected
with 4M HCl in dioxane to form the free hydrazide group.
Lipid-PEG-hydrazide may then be incorporated into liposomes. The
hydrazide groups are reactive towards aldehydes, which as described
above, can be generated on numerous biologically relevant
compounds.
[0086] The methods just described may be applied to a variety of
lipid amines, including PE, cholesteryl amine, and glycolipids with
sugar amine groups. It will be appreciated that a variety of
alternative coupling reactions, in addition to those just
described, are suitable for preparing vesicle-forming lipids
derivatized with hydrophilic polymers such as PEG, having terminal
groups which are activated or are reactive in protein coupling
reactions.
[0087] 1. Maleimide Coupling. Maleimides are widely used protein
modifying reagents and are especially useful when the maleimide is
one of two functional groups in a heterobifunctional crosslinking
reagent. The reaction of maleimides with sulfhydryl groups involves
Michael addition of the mercaptan group to the activated double
bond. Reaction with amino groups occurs by the same mechanism, but
at a much slower rate. Since mercaptan is the most reactive
species, particularly at neutral pH, the maleimide group can be
used to target a small number of sulfhydryl groups and good
selectivity is usually achieved.
[0088] In one preferred embodiment, a derivatized lipid, such as
PE- or DSPE-PEG, is prepared to contain a terminal maleimide group
(compounds VI and XVI), as illustrated in FIGS. 1 and 4 above. The
lipid, after incorporation into liposomes, is then reacted with a
sulfhydryl-containing effector, typically a polypeptide, under
suitable coupling conditions. The reaction of the terminal
maleimide-PEG lipid (compound VI or XVI) with a peptide sulfhydryl
group is illustrated in FIG. 9. As shown, the reaction couples the
protein to the lipid polymer through a thioether linkage, to give
the derivatized DSPE (compound XXXIII). Use of this synthetic
approach to couple proteins to liposomes is described in Example
6.
[0089] The efficiency of .beta.-galactosidase coupling to liposomes
containing a maleimide coupling agent in the presence or absence of
DSPE-PEG3500 has been examined and the results are discussed
below.
[0090] Reactions were carried out with liposomes prepared to
contain, as the maleimide coupling agent, either (a) the DSPE
derivative of succinimidyl 4-(p-maleimidophenyl)butyrate (MBP), (b)
the DSPE derivative of N-(11-maleimido-undecanoyl) (C11), or (c)
the maleimide of PE-PEG3500. Reactions carried out with (c) are
described in detail in Example 6.
[0091] After carrying out the protein-liposome coupling reaction,
performed as described above for (a)-(c), the amount of
liposome-bound enzyme was quantitated. Recovery of liposomes was
measured by scintillation counting and the recovery of protein was
measured by the beta-galactosidase assay and direct quantitation of
the protein amount as described in Example 6.
[0092] The maleimide of the DSPE carbamide of PEG3500 was very
effective in crosslinking .beta.-galactosidase to liposomes, either
in the presence or absence of DSPE-PEG3500 chains. As seen in Table
2, there was essentially no difference in the amount of protein
crosslinked to either type of liposome in two separate experiments.
In addition, the amount of protein coupled to the PE-PEG maleimide
was much higher than to either the MPB or MPB-C.sub.11
maleimides.
[0093] The presence of "non-activated" DSPE-PEG3500 in the
liposomes had little effect on the levels of coupling of the
protein to DSPE-PEG-maleimide liposomes, but inhibited the level of
protein coupling to liposomes containing either the MPB lipid, or
the MBP-C.sub.11 lipid. TABLE-US-00002 TABLE 2 Phenotype'' PEG-DSPE
Crosslinker 10 mM 2-ME ng Protein/.mu.mol Lipid* - MPB 1609/2284 -
MPB + (-80) + MPB (-282) - C.sub.11 690 - C.sub.11 + 847 + C.sub.11
358 (-157) + C.sub.11 + 80 - 3500 10,033 - 3500 + 572 + 3500
10,765/12,412 + 3500 + 110 *Background binding in the absence of
crosslinker has been subtracted. Background values range from
500-1000 ng protein/.mu.mol lipid. There was a tendency for
background values to be somewhat (10-30%) higher in the presence of
PEG-DSPE; this may not be significant. Multiple entries denote
multiple separate crosslinking experiments.
[0094] 2. Coupling by 3-(2-pyridyldithio)propionamide.
[0095] The reaction of dithio propionamides with a sulfhydryl group
produces coupling of functionalized lipids to sulfhydryl-containing
molecules via a disulfide linkage. Disulfide exchange occurs
readily at pH 8, in a non-reducing environment. The method involves
reaction of a thiol group in a peptide with a liposome prepared to
contain DSPE-PEG-(2-pyridyldithio) propionamide). The reaction
couples the protein to the liposomes through a disulfide linkage as
illustrated in FIG. 10 (compound XXXIV).
[0096] 3. Reductive Amination
[0097] In this approach, the terminal hydroxyl group of a PEG
chain, covalently linked at one end to PE or DSPE, is converted to
the corresponding aldehyde by oxidation under mild conditions. The
oxidation step may be carried out before or after incorporation
into liposomes to produce the aldehyde form of the derivatized
lipid (compound XIII in FIG. 3). Reaction of the aldehyde with the
amine group of an effector molecule gives the Schiff base (compound
XXXV, FIG. 11) which is then reduced to the desired derivatized
lipid containing an amino-linked peptide (XXXVI).
[0098] As indicated above, the polymers can also be activated for
effector coupling in preformed lipids, i.e., with the
polymer-derivatized lipids already incorporated into liposomes. One
advantage of this approach is that only polymer moieties on the
outer surface of the liposomes are activated. In one general
approach involving PEG polymers, the terminal OH groups are first
oxidized by treatment with sodium periodate for 2 hours at
20.degree. C. in the dark. After oxidation, the excess reagent is
removed, and the liposomes are incubated with the effector
molecule, e.g. F.sub.ab fragments, in the presence of 2M sodium
cyanoborohydride (10 .mu.L/mL) at 20.degree. C. for 14 hours. After
completing the incubation, the liposomes can be chromatographed on
a Sepharose to remove free (non-linked) effector molecules.
III. Bloodstream and Tissue Retention of Liposomes Containing
End-Functionalized PEG-DSPE
[0099] In vivo studies were undertaken to determine the bloodstream
and tissue retention of liposomes containing end-functionalized
PEG-DSPE, as described in Example 7. End-functionalized PEG-DSPE
contains a chemically active group which can be used for attaching
a variety of compounds to liposomes. From these studies it has been
determined that end-functionalization does not affect the extended
lifetime in the bloodstream of liposomes containing PEG-DSPE,
monomethoxy PEG-DSPE, or other similarly modified vesicle-forming
lipids.
[0100] In experiments performed in support of the present
invention, liposomes containing PEG-DSPE end-functionalized by
hydrazide were prepared. The hydrazide group at the end of a PEG
chain can be used for the introduction of other functional groups,
or can be used in numerous types of conjugation schemes (Inman, J.
K., Meth. Enzymol. 34:30-58 (1974)). Particularly useful is
hydrazide's reactivity toward various glycoproteins, such as
immunoglobulins (Wilchek, M., and Bayer, E. A., Meth. Enzymol.
138:429-442 (1987)), for attaching these molecules to
liposomes.
[0101] Gallium 67-labelled, hydrazide end-functionalized PEG
liposomes were injected in rats by tail vein injection at about
10-20 micromolar phospholipid/kg body weight. Blood samples were
obtained by retroobital bleeding at defined times. The percent of
gallium labelled liposomes remaining in the bloodstream was
determined at 0, 15 minutes, 1, 3, 5, and 24 hours and is presented
in Table 3. The percent injected gallium 67-labelled liposome dose
remaining in the blood stream at different times is illustrated in
a half log plot versus time in FIG. 12.
[0102] After 24 hours the animals were sacrificed and tissues
removed for label quantitation. The percent of the injected dose
found in selected tissues at 24 hours is presented in Table 3.
[0103] The blood and tissue retention of Ga-labelled, hydrazide
end-functionalized liposomes having two different lipid
compositions were also compared as shown in Table 3. A fluid
liposome composition was prepared from partially hydrogenated egg
phosphatidylcholine (PHEPC). A typical liposome composition
contains the hydrazide PEG-DSPE lipid, partially hydrogenated egg
PC (PHEPC), and cholesterol in a lipid:lipid:lipid mole ratio of
about 0.15:1.85:1. A rigid liposome composition was prepared by
substituting hydrogenated serum phosphatidylcholine (HSPC) for
PHEPC at the same mole ratio.
[0104] As is indicated in Table 3, the fluidity of the liposome
composition does not affect the blood retention time of the
liposomes. However, the fluidity of the liposome composition does
appear to affect the tissue distribution of the end-functionalized
liposome. For example, rigid liposomes are preferentially retained
by liver, spleen and bone tissue. Fluid liposomes are
preferentially retained by the kidneys, heart, skin and muscle
tissue. TABLE-US-00003 TABLE 3 % Injected 67 GA Dose Detected at
Specified Timepoints Peg-HZ Rigid PEG-Hz Fluid Blood 0 101.1 .+-.
12.0 100.2 .+-. 5.4 15 min. 89.6 .+-. 11.2 81.6 .+-. 2.5 1 hr. 84
.+-. 11.1 81.7 .+-. 7.4 3 hr. 76 .+-. 10.5 75.3 .+-. 5.1 5 hr. 71.7
.+-. 10.7 66.3 .+-. 3.8 24 hr. 33.4 .+-. 6.8 34.3 .+-. 0.68 Tissues
at 24 hr. liver 12.1 .+-. 1.2 8.8 .+-. 0.81 spleen 5.1 .+-. 0.47
4.7 .+-. 0.64 kidneys 1.4 .+-. 0.22 1.7 .+-. 0.25 heart 0.36 .+-.
0.037 0.77 .+-. 0.21 lungs .62 .+-. 0.23 0.58 .+-. 0.03 skin .086
.+-. 0.03 0.16 .+-. 0.08 muscle .08 .+-. 0.03 0.29 .+-. 0.02 bone
.28 .+-. 0.09 0.04 .+-. 0.01
IV. Therapeutic Effector Compositions
[0105] Below are described specific embodiments of the effector
composition of the invention, and their intended use as injectable
therapeutic agents.
[0106] A. Compositions for Enhancing an Immune Response
[0107] In one general embodiment, the effector in the liposome
composition is a molecule capable of enhancing an immune response
when administered parenterally.
[0108] 1. F.sub.ab Effector. The F.sub.ab effector composition is
used as a passive vaccine to provide humoral immunity against one
of a variety of selected pathogenic antigens. The composition is
administered to supplement a weakened immune response to a given
antigen.
[0109] The vaccine effector composition is administered
intravenously shortly after exposure to, or shortly before expected
exposure to a selected pathogen. The composition is preferably
injected in an amount corresponding to between about 0.1 to 2 mg
antibody/kg body weight. After IV administration, the composition
circulates in the bloodstream, at an effective concentration, for
1-2 days.
[0110] 2. CD4 Glycoprotein Effector. Numerous therapies for the
prevention and treatment of human immunodeficiency virus (HIV)
infection and acquired immune deficiency syndrome (AIDS) have been
proposed. These therapies target different steps in the process of
viral infection. Frequently, therapy includes the administration of
drugs which interfere with viral replication, such as AZT and DDI.
The administration of these drugs is accompanied by toxic side
effects, since the replication process of normal cells is also
affected.
[0111] Another step in the process of viral infection which is
targeted in therapy is viral attachment to cells. HIV binds with
specificity to the CD4 receptor of CD4+ T cells. By mechanisms not
yet fully understood, the CD4+ cells eventually can become infected
by HIV. Soluble CD4 receptor polypeptides have been administered
intravenously to HIV-infected patients to prevent further HIV
infection of a patient's CD4+ T cell population. Heretofore, this
therapy has not been effective, since CD4 receptor fragments are
rapidly cleared from circulation in the blood stream, and
inhibitory plasma concentrations cannot be maintained (Capon and
Ward).
[0112] The effector molecule in this embodiment is a soluble CD4
receptor polypeptide capable of binding to the gp120 glycoprotein
of human immunodeficiency virus (HIV) to prevent binding of HIV to
CD4+ T cells. In a preferred embodiment covalent attachment of CD4
is accomplished by coupling periodate oxidized CD4 with hydrazide
group containing liposomes.
[0113] CD4 administered as a long-circulating liposomal composition
will remain in the blood stream for a longer period of time. The
CD4 effector composition can be administered intravenously during
early or late stages of HIV infection, most beneficially in
combination with other drugs used in AIDS therapeutics, so that HIV
particles bound to the liposomes, to the extent these are taken up
by infected cells, will also deliver a dose of the anti-viral agent
to the infected cells. AZT and DDI are examples of anti-HIV drugs
which may be encapsulated in the liposome compositions.
[0114] The liposome composition should be administered
intravenously in a dose equivalent to an effective blood stream CD4
concentration of 1-10 micromolar. Doses of 5-40 mg CD4/kg body
weight can be administered, typically at intervals of 2-14 days
between treatments, with the level of HIV present in the
bloodstream being monitored during treatment by standard assay
methods.
[0115] Principal advantages of this composition are the increased
circulation time of the CD4 effector in the blood stream and the
polyvalent presentation of the effector on the surface of the
liposomes. Improved affinities of polyvalent CD4 presentation has
recently been described (Chen, L. L., et al., J. Biol. Chem.
266:18237-18243 (1991)). As described above, CD4 receptor fragments
are cleared rapidly by renal filtration. Covalent attachment of the
CD4 polypeptide to liposomal carriers prevents renal clearance, and
permits circulation of the polypeptide effector composition for
24-48 hours in the blood stream.
[0116] Additionally, the polyvalent CD4-bearing liposomes resemble
CD4+ T cell lymphocytes in that the CD4 glycoproteins are presented
on hydrophobic surfaces which mimic the surfaces of T cell
lymphocytes. This presentation is likely to serve as a decoy
binding HIV particles and HIV infected cells expressing gp120 so
that healthy CD4+ lymphocytes are spared.
[0117] 3. Effector for Stimulating Inflammatory Immune
Responses.
[0118] Some medical conditions are treated indirectly, by
stimulation of the body's natural immune response. Such conditions
can include immunodeficiency diseases, such as AIDS, chronic
infectious, and certain types of cancers. One immunostimulant
therapy involves intravenous injection of cytokines, which can acts
to stimulate B cell and T cell immune responses in a variety of
ways.
[0119] The cytokine effector composition may be administered on a
short term basis to enhance a weak immunogenic or weak microbicidal
response. Alternatively, the cytokine effector composition may also
be administered on a long term basis as part of a therapy treatment
for cancer or AIDS. The effector composition may be administered
intravenously at doses of about 0.5 to 5.0 mg/kg body weight to
enhance an immunogenic response. These doses result in an effective
cytokine concentration of about 0.1-1 micromolar in the blood
stream.
[0120] B. Compositions for Blocking Binding to Cell Receptors
[0121] In another general embodiment, the effector in the liposome
composition is a molecule capable of blocking the binding of an
endogenous agent to a cell receptor, to achieve a desired
therapeutic effect.
[0122] 1. ELAM-1 Binding Inhibitor. As one example, inflammation
causes the expression of a polypeptide, endothelial leukocyte
adhesion molecule-1 (ELAM-1), on the surface of endothelial cells
of the blood vessels. ELAM-1, in turn, recognizes and binds a
polysaccharide moiety on surfaces of neutrophils, and recruits
neutrophils to sites of inflammation. By preventing the recognition
and binding of neutrophils by ELAM-1, excessive inflammatory
responses due to conditions, such as reperfusion injury, septic
shock, and chronic inflammatory diseases, can be avoided.
[0123] In this embodiment, the effector is used to prevent the
excessive recruitment of neutrophils to sites of inflammation in
the blood stream. The effector is sialyl Lewis.sup.x recognized by
ELAM-1 (Phillips). This polysaccharide effector is covalently
attached to long-circulating liposomal compositions by the methods
described above. In a preferred embodiment attachment of sialyl
Lewis.sup.x to liposomes is accomplished via the reducing end of
the glucosamine residue. The reducing end can easily react with a
hydrazide group of a DSPE-PEG preparation. Coupling of the
polysaccharide to the liposomal carrier composition prevents the
polysaccharide's clearance by the kidney, and maintains an
effective concentration of the polysaccharide effector over a 48
hour period. The liposomal carrier composition is administered in
doses of 10 to 50 micrograms/ kg body weight in a timely fashion,
intravenously, and close to the site of inflammation.
[0124] 2. Inhibitor of IL-1 Activity. As a second example, the
effector is IL-1 inhibitor, which inhibits IL-1's immunostimulatory
activity, or IL-1 receptor antagonist (IL1RA), which blocks the
binding of IL-1 to lymphocyte cell surfaces. These molecules may be
administered to a subject for treatment of septic shock, toxic
shock, colonic inflammation, or leukemic cell proliferation. In
this aspect of the invention, the liposomal carrier composition is
administered in doses of 20 to 50 micrograms/kg body weight on a
short term basis for the treatment sepsis, toxic shock or colonic
inflammation. The liposomal carrier composition may also be
administered at 1 to 2 day intervals on a long term basis for the
treatment of leukemia.
[0125] Other molecules effective to block the binding of specific
cytokines to specific lymphocyte populations may also be
employed.
[0126] The use of the long-circulating effector composition, for
use in blocking the binding of endogenous agents to cell receptor
sites, provides two advantages over the use of free effector.
First, the effector is maintained in the bloodstream over an
extended period, by virtue of blocking renal clearance of the
effector. Secondly, the effector molecule, in liposome-bound form,
provides greater steric hindrance at the cell surface site of the
receptor. Also, the competitive binding or blocking effector and
endogenous agent on the receptor site is shifted toward the
blocking agent, since the liposomal structure will be displaced at
a slower rate kinetically, due to its larger size and number of
blocking molecules in the region of the receptor site.
[0127] 3. Peptide Hormones. In this embodiment the effector
composition is useful in the treatment of various diseases that
respond to peptide hormones. In one embodiment, the effector is
parathyroid hormone (PTH) which is effective to inhibit
uncontrolled osteoblast division.
[0128] 4. Peptide. In this embodiment, the effector is a short
peptide that has cell-binding activity and is effective to compete
with a ligand for a receptor site. Inhibition of the
ligand-receptor cell-binding event potentially results in arresting
the infection process.
[0129] In general, useful peptides will have cell-binding activity
due to a portion of sequence other than the end of the peptide. In
this way, after attachment to the polymer chain on the liposome,
the peptide remains active. Another general feature of useful
peptides is their small size. Peptides of between about 4-20 amino
acids are preferred.
[0130] One exemplary peptide, YIGSR, identified herein as SEQ ID
NO:6 (FIG. 13), is useful for blocking metastases of tumors. SEQ ID
NO:6 is one of the peptide sequences in the B1 chain of laminin
responsible for the glycoprotein's adhesive properties and is known
to bind to the laminin receptor. Laminin, the protein in which the
YIGSR sequence occurs, is a constituent of basement membranes.
Circulating metastatic cells which over-express the laminin
receptor may find their way to laminin molecules in the basement
membrane where they may become attached and establish metastatic
tumors. By introducing exogenous YIGSR, the laminin receptors of
circulating metastatic cells are blocked, thereby inhibiting tumor
establishment.
[0131] Similarly, the peptide arginine-glycine-aspartic acid-serine
(RGDS) has experimentally been shown to inhibit the establishment
of metastatic tumors by interfering with the binding of tumor cells
to fibronectin (Humphries, M. J., et al., Science 233:467-469
(1986)). Like YIGSR, RGDS is a peptide sequence involved with tumor
cell adhesion to basement membranes.
[0132] The infection of lymphocytes by HIV also involves a specific
peptide-receptor interaction (Nehete, P. N., et al., J. Virol.
67:6841-6846 (1993)). Here, the receptor is the CD4 protein and the
peptide is the HIV envelope protein gp120. The peptide binding
sequences are located in the V3 loop of gp120. Several peptide
sequences of between 8-15 amino acids have been implicated in the
binding interactions. These sequences include SEQ ID NO:1 through
SEQ ID NO:5 and are shown in FIG. 13.
[0133] Pseudomonas cepacia infections also exhibit specific binding
to the cells they infect (Sajjan, U. S., et al., Inf. Immun.
61:3157-3163 (1993)). Pseudomonas pilin proteins, which are found
on the bacterial cell surface, act as receptors for host proteins
called mucins. Suitable peptides have been disclosed (e.g., Sastry,
P. A., et al., J. Bacteriology 164(2):571-577 (1985); Lee, K. K.,
et al., Molecular Microbiol. 3(11):1493 (1989)).
[0134] C. Antimicrobial Composition
[0135] In this embodiment the effector is a compound which is
useful in the prevention and treatment of septic shock. The causal
agents of septic shock are endotoxins which accumulate during
systemic gram-negative bacterial infections (Jawetz, E., in BASIC
AND CLINICAL PHARMACOLOGY (Katzung, B. G., Ed.) Apple & Lange,
Los Altos, Calif., pg. 511 (1987)). Because of the rapid onset of
severe sepsis, treatment is often not begun until critical stages
of sepsis.
[0136] The antimicrobial agent which has been used most
successfully in treating septic shock against in cases of septic
shock is polymyxin B. Because the compound is rapidly excreted,
high doses of polymyxin B are required for effective treatment. The
high doses, unfortunately, can lead to severe renal toxicity.
[0137] In the present invention, polymyxin B circulation in the
bloodstream is extended severalfold by its attachment to
long-circulating liposomes. The compound is attached to
long-circulating liposomal composition carriers by the coupling
methods described above.
[0138] The liposomal composition is administered on a short term
basis, at a dose of 0.1-0.5 mg/ kg body weight, as a prophylactic
for individuals at risk of, or suffering from acute septic shock.
Features of the polymyxin B liposomal composition, already
discussed, will minimize polymyxin B's renal accumulation and renal
toxicity.
[0139] The following examples illustrate methods for preparing
derivatized lipids and protein-coated liposomes in accordance with
the invention.
EXAMPLE 1
Preparation of DSPE-PEG-Maleimide
[0140] A. Preparation of the Mono 2-nitrobenzene-sulfonamide of PEG
bis(amine) (compound II)
[0141] A mixture of 1.7 g (0.5 mmole) of commercially available
polyethylene glycol bis(amine) and 104 mg (0.55 mmole) of
2-nitrobenzene sulfonyl chloride were added to a round-bottomed
flask. The minimum amount of dioxane to effect solution (about 15
mL) and 280 microliters of triethylamine (2 mmole) were added. The
reaction flask was stoppered and allowed to stand at room
temperature for 4 days.
[0142] Thin layer chromatography (TLC) on silica coated plates
using a solvent mixture of the following composition
CHCl.sub.3:CH.sub.3OH:H.sub.2O:NH.sub.4OH, 130:70:8:0.5 (v/v/v/v),
showed fluorescence quenching spots at R.sub.f=0.87 to 0.95 and
R.sub.f=0.68-0.75. The 2-nitro benzene sulfonyl chloride was a more
compact spot at R.sub.f=0.85. The UV absorbing material at
R.sub.f=0.87-0.95 was tentatively identified as the
bis-2-nitro-benzenesulfenamide. The material at R.sub.f=0.68-0.75
was assigned to the desired mono-2-nitrobenzenesulfonamide of the
starting diamine.
[0143] The solvent was evaporated under vacuum to obtain 2.135 g of
a yellow syrup. The crude syrup was dissolved in 5 mL chloroform
and placed at the top of a 21 mm.times.270 mm column of SiO.sub.2
wetted with chloroform. The product was purified by passing through
the column, in sequence: TABLE-US-00004 Volume % Volume % MeOH
containing Amount (mL) Chloroform 1% conc. NH.sub.4OH 100 100% 0%
200 90% 10% 100 80% 20% 100 70% 30%
[0144] Fifty mL aliquots were collected separately and assayed by
TLC as described above. Most of the yellow, ninhydrin
positive-reacting material was eluted in the 20% MeOH fraction. The
fractions were dried and resulted in recovery of 397 mg of a bright
yellow solid. The yield of the pure sample was about 20%.
[0145] B. Preparation of the Imidazole Carbamide of the Mono
2-nitrobenzenesulfonamide of PEG bis(amine)(compound III)
[0146] 550 mg (0.15 mmole) of the 2-nitrobenzenesulfonamide of PEG
bis(amine), compound II, were dissolved in anhydrous benzene. To
this was added 49 mg of carbonyl diimidazole (0.3 mmole) and 28
microliters (0.20 mmole) of triethylamine. The air in the reaction
vessel was displaced with nitrogen, the flask sealed and the
reaction mixture was heated in an 80.degree. C. oil bath for 4
hours. TLC on silica-coated plates using the same solvent system as
described above showed that all of the starting sulfonamide
(Rf=0.72) had been consumed, and had been replaced by an iodine
absorbing material at Rf=0.92. The solvent was removed under
vacuum. The residue was dissolved in about 2.5 mL chloroform and
transferred to the top of a 21.times.280 mm column of silica which
was wetted with chloroform. The following solvents were passed
through the column, in sequence: TABLE-US-00005 Volume % Volume %
MeOH containing Amount (mL) Chloroform 1% conc. NH.sub.4OH 100 100%
0% 100 90% 10% 200 80% 20%
[0147] 50 mL fractions were collected and assayed by TLC. The
desired product, compound III, was found predominantly in the 80-20
chloroform-methanol fractions. Upon evaporating the pooled
fractions to dryness, 475 mg of a lemon-yellow solid was obtained
(compound III).
[0148] C. Preparation of the DSPE Carbamide of the 2-Nitrobenzene
Sulfonamide of PEG Bis(amine)
[0149] To the 450 mg (0.125 mmole) of 2-nitrobenzenesulfonamide of
the imidazole carbamide of PEG bis(amine) (compound III) dissolved
in 4.5 mL benzene was added 93 mg DSPE (0.125 mmole) and 70
microliters (0.50 mmole) of triethylamine. The reaction flask was
then flushed with nitrogen, stoppered, and the contents heated in
an oil bath at 80.degree. C. for 6 hours with stirring. The
reaction mixture was then cooled to room temperature and analyzed
by TLC. TLC indicated that all of the DSPE had been consumed (e.g.,
the reaction had gone to completion). The solvent was evaporated
under vacuum and the residue was dissolved in 2.5 mL chloroform and
placed at the top of a 21.times.260 mm column of silica wetted with
chloroform. The sample was purified by passing through the column
in sequence: TABLE-US-00006 Volume % Volume % MeOH containing
Amount (mL) Chloroform 1% conc. NH.sub.4OH 100 100% 0% 200 90% 10%
100 80% 20% 100 70% 30%
[0150] The desired product eluted at 20% (1% conc. NH.sub.4OH in
MeOH), was evaporated and afforded 358 mg of a bright yellow solid
with an Rf=0.95. Fractions containing imidazole were not used and
the final yield of the product (0.0837 mmoles) was 65%.
[0151] D. Preparation of the DSPE Carbamide of PEG Bis(amine)
(Compound IV)
[0152] The product from Example 1C above (.about.358 mg) was
dissolved in 10 mL ethanol. To this solution was added 2.4 mL water
and 1.2 mL acetic acid. The mixture was allowed to stand at room
temperature for 18 hours. TLC analysis after 18 hours indicated
that only partial deprotection had occurred. To the reaction
mixture was added another 2.3 mL water and 1.2 mL acetic acid and
the reaction mixture was then allowed to stir overnight. TLC
analysis on silica-coated plates using a similar solvent system as
described above revealed florescence quenching materials with
R.sub.f values of 0.86 and 0.74, respectively. The desired
ninhydrin reactive, phosphate-containing material migrated with an
Rf value of 0.637. This spot showed no fluorescence quenching.
[0153] The solvent was removed under vacuum. The remaining residue
was redissolved in 15 mL chloroform and extracted with 15 mL 5%
sodium carbonate. The mixture was centrifuged to effect separation,
and the sodium carbonate phase was reextracted 2.times. with 15 mL
chloroform. The combined chloroform extracts were evaporated under
reduced pressure to obtain 386 mg of wax. TLC indicated that the
wax was largely a ninhydrin positive, phosphate containing lipid of
R.sub.f=0.72.
[0154] The wax was dissolved in 2.5 mL chloroform and placed on a
silica column which had been wetted with chloroform. The following
solvents were passed through the column in sequence: TABLE-US-00007
Volume % Volume % MeOH containing Amount (mL) Chloroform 1% conc.
NH.sub.4OH 100 100% 0% 200 90% 10% 100 80% 20% 100 70% 30% 100 50%
50% 100 0% 100%
[0155] The samples were assayed by TLC. The desired product was
found in fractions containing 70-30 and 50-50 chloroform-methanol
as eluent. These samples were combined and evaporated to dryness
under vacuum to afford 91 mg (22 micromoles) of a viscous
syrup.
[0156] E. Preparation of the Maleic Acid Derivative of the DSPE
Carbamide of PEG Bis(amine) (Compound V)
[0157] To 18 micromoles of the viscous syrup prepared in Example 1D
above and dissolved in 1.8 mL chloroform was added 3.5 mg (36
micromoles) maleic anhydride and 5 microliters (36 micromoles)
triethylamine. The stoppered flask containing the reaction mixture
was allowed to stand at room temperature for 24 hours and the
solvent was subsequently evaporated under reduced pressure. TLC on
silica plates indicated that all of the starting material had been
replaced by a ninhydrin-negative, phosphate containing material of
Rf=0.79-1.00 (Compound V).
[0158] F. Preparation of the Maleimide of the DSPE Carbamide of PEG
Bis(amine) (Compound VI)
[0159] The syrup was dissolved in 2 mLs acetic anhydride saturated
with anhydrous sodium acetate. The solution was heated in a
50.degree. C. oil bath for two hours. After cooling to room
temperature, 10 mL ethanol was added to the contents of the flask
and the volatile components were then evaporated under vacuum. This
step was repeated twice to remove excess acetic anhydride and
acetic acid. The resulting residue was taken up 1 mL chloroform and
passed through a silica gel column using the following solvents in
sequence: TABLE-US-00008 Volume % Volume % MeOH containing Amount
(mL) Chloroform 1% conc. NH.sub.4OH 100 100% 0% 200 90% 10% 100 80%
20% 100 70% 30%
[0160] 50 mL samples were collected and the main product was found
in the fractions eluted with 90-10 chloroform-methanol. The
fractions were combined and evaporated to dryness under vacuum to
afford 52 mg of a pale yellow viscous oil, which by TLC migrated
with an Rf of 0.98 and was determined to contain phosphate. 12.3
micromoles of product (compound VI) were obtained, corresponding to
a yield of about 34%.
EXAMPLE 2
Preparation DSPE-PEG 3-(2-pyridyldithio)propionamide
[0161] The DSPE carbamide of PEG bis(amine) (compound IV, 50
micromoles) is dissolved in 3 mL of anhydrous methanol containing
50 micromoles of triethylamine and 25 mg of N-succinimidyl
3-(2-pyridyldithio)propionate (SPDP, Pierce, Rockford, Ill.). The
reaction is carried out at room temperature for 5 hours under an
argon atmosphere. Methanol is removed under reduced pressure, and
the products are redissolved in chloroform and applied to a 10 mL
silica gel column, using silica gel which has been previously
activated at 150.degree. C. overnight. A similar solvent system as
described in Example 1 is used to purify the product. Analysis on
TLC plates indicates a product (compound VIII) with an R.sub.f=0.98
which reacts negatively with ninhydrin, contains phosphate and has
no free sulfhydryl groups. When the product is treated with excess
dithiothreitol, 2-thiopyridinone is released.
EXAMPLE 3
Preparation of a PEG-Derivatized PE Containing a Terminal Aldehyde
Group
[0162] A. Preparation of 1-trimethylsilyloxy-PEG (Compound X)
[0163] 15.0 gm (10 mmoles) of PEG MW 1500, (Aldrich Chemical, St.
Louis, Mo.) was dissolved in 80 mL benzene. 1.40 mL (11 mmoles) of
chlorotrimethyl silane (Aldrich Chemical Co.) and 1.53 mL (1
mmoles) of triethylamine was added. The mixture was stirred at room
temperature under an inert atmosphere for 5 hours.
[0164] The mixture was filtered by suction to separate crystals of
triethylammonium chloride and the crystals were washed with 5 mL
benzene. Filtrate and benzene wash liquids were combined. This
solution was evaporated to dryness under vacuum to provide 15.83
grams of colorless oil which solidified on standing.
[0165] TLC of the product on Si-C.sub.18 reversed-phase plates
using a mixture of 4 volumes of ethanol with 1 volume of water as
developer, and iodine vapor visualization, revealed that all the
polyglycol 1500 (R.sub.f=0.93) had been consumed and was replaced
by a material of R.sub.f=0.82. An infra-red spectrum revealed
absorption peaks characteristic only of polyglycols.
[0166] Yield of 1-trimethylsilyloxy-PEG, M.W. 1500 (compound X) was
nearly quantitative.
[0167] B. Preparation of Trifluoromethane Sulfonyl Ester of
Trimethylsilyloxy-PEG (Compound XI)
[0168] 15.74 grams (10 mmol) of the crystalline 1-trimethylsilyloxy
PEG obtained as described above (compound X) was dissolved in 40 mL
anhydrous benzene and cooled in a bath of crushed ice. 1.53 mL (11
mmol) triethylamine and 1.85 mL (11 mmol) of
trifluoromethanesulfonic anhydride obtained from Aldrich Chemical
Co. were added and the mixture was stirred overnight under an inert
atmosphere until the reaction mixture changed to a brown color.
[0169] The solvent was then evaporated under reduced pressure and
the residual syrupy paste was diluted to 100.0 mL with methylene
chloride. Due to the reactivity of trifluoromethane sulfonic
esters, no further purification of the trifluoromethane sulfonyl
ester of 1-trimethylsilyloxy PEG carried out.
[0170] C. Preparation of 1-Trimethylsilyloxy PEG 1500 PE
Intermediate (Compound XII)
[0171] 10 mL of the methylene chloride stock solution of the
trifluoromethane sulfonyl ester of 1-trimethylsilyloxy PEG
(compound XI) was evaporated to dryness under vacuum to obtain
about 1.2 grams of residue (approximately 0.7 mmoles). To this
residue, 3.72 mL of a chloroform solution containing 372 mg (0.5
mmoles) egg PE was added. To the resulting solution, 139
microliters (1.0 mmole) of triethylamine was added and the solvent
was evaporated under vacuum. To the residue was added 5 mL dry
dimethyl formamide and 70 microliters (0.50 mmoles) triethylamine
(VI). Air from the reaction vessel was displaced with nitrogen. The
vessel was sealed and heated in a sand bath at 110.degree. C. for
22 hours. The solvent was evaporated under vacuum to obtain 1.58
grams of brownish colored oil.
[0172] A 21.times.260 mm column filled with Kieselgel 60 silica
gel, 70-230 mesh, was prepared and wetted with a solvent composed
of 40 volumes of butanone, 25 volumes acetic acid and 5 volumes of
water. The crude product was dissolved in 3 mL of the same solvent
and chromatographed using the above-described solvent system.
Sequential 30 mL portions of effluent were each assayed by TLC.
[0173] The TLC analysis was carried out on silica gel coated glass
plates using a solvent combination of butanone/acetic acid/water;
40/25/5; v/v/v. Visualization was carried out using iodine vapor
absorption. In this solvent system, N-1-trimethylsilyloxy
PEG-1500-PE appeared at R.sub.f=0.78, Unreacted PE appeared at
R.sub.f=0.68.
[0174] The desired N-1-trimethylsilyloxy PEG 1500 PE was a chief
constituent of the 170-300 mL portions of column effluent. When
combined and evaporated to dryness under vacuum, these portions
afforded 111 mg of a pale yellow oil
(1-trimethylsilyloxy-PEG-1500-PE intermediate).
[0175] D. Preparation of Polyethylene Glycol 1500: PE (Compound
XII)
[0176] Once-chromatographed, the trimethylsilyloxy intermediate
from Example 3C above was dissolved in 2 mL of tetrahydrofuran. To
this, 6 mL acetic acid and 2 mL water was added. The resulting
solution was allowed to stand for 3 days at 23.degree. C. The
solvent from the reaction mixture was evaporated under vacuum and
the resulting residue was dried to constant weight to obtain 75 mg
of pale yellow wax. TLC on Si-C18 reversed-phase plates eluted with
a solvent mixture of 4:1 ethanol-water (v/v) indicated that some
free PE and some polyglycol-like material formed during the
hydrolysis.
[0177] The residue was dissolved in 0.5 mL tetrahydrofuran and
diluted with 3 mL of a solution of 80:20 ethanol:water (v/v). The
solution was applied to the top of a 10 mm.times.250 mm
chromatographic column packed with octadecyl bonded phase silica
gel and the crude product was eluted with an 80:20 ethanol:water
(v/v) solvent system, collecting sequential 20 mL portions of
effluent. The effluent was assayed by reversed phase TLC. Fractions
containing product (Rf=0.08 to 0.15) were combined. When evaporated
to dryness under vacuum these portions afforded 33 mg of a
colorless wax (compound XII) corresponding to a yield of only 3%,
based on the starting phosphatidyl ethanolamine.
[0178] NMR analysis indicated that the product incorporated both PE
residues and PEG residues. The product was used to prepare PEG-PE
liposomes.
[0179] E. Preparation of the Aldehyde of PEG-PE (Compound XIII)
[0180] The free hydroxyl group on PEG derivatized PE (compound XII)
can be oxidized to the corresponding aldehyde in the following
manner (Harris, J. M., J. Polym. Sci., Polym. Chem. Ed. 22:341-352
(1984)) prior to incorporation of the functionalized polymers into
liposomes. About 2.7 g PEG1500-PE (1 mmole), prepared as in Example
3D, is added to 0.4 g acetic anhydride in 15 mL dimethylsulfoxide
and the resulting mixture is stirred for 30 hours at room
temperature. The reaction mixture is then neutralized by addition
of dilute sodium hydroxide and the solvent is evaporated under
reduced pressure to yield a sticky residue.
[0181] The progress of the reaction may optionally be monitored by
withdrawing aliquots of the reaction mixture, performing a mini
work-up as described below, and monitoring the appearance of an IR
absorption corresponding to an aldehyde group.
[0182] The sticky residue is dissolved in 10 mL chloroform, washed
with two successive 10 mL portions of water, and the organic phase
is dried over a drying agent such as anhydrous magnesium sulfate.
The product-containing chloroform phase is evaporated under vacuum
to obtain a wax. The wax is then redissolved in 5 mL chloroform and
purified by column chromatography on silica gel using the following
series of solvents: TABLE-US-00009 Volume % Volume % Methanol
containing 2% Chloroform Conc. Ammonium Hydroxide/Methanol 100% 0%
95% 5% 90% 10% 85% 15% 80% 20% 70% 30% 60% 40% 50% 50% 0% 100%
[0183] Typically, 50 mL fractions of column effluent are collected
and analyzed by TLC on Si-C18 reversed-phase plates using a 4:1
ethanol:water (v/v) solvent system followed by I.sub.2-vapor
visualization.
[0184] Only those fractions containing an iodine-absorbing lipid
with an Rf value of about 0.20 are combined and evaporated to
dryness under vacuum, followed by drying under high vacuum to
constant weight to yield 94 mg of a waxy crystalline solid product
(compound XIII) with a molecular weight of 2226.
EXAMPLE 4
Synthesis of N-hydroxysuccinimide ester of
.alpha.-hydroxy-.omega.-(carboxymethylamino-carbonyl) PEG (Compound
XXIV) and Coupling to DSPE
[0185] An .alpha.-hydroxy-.omega.-carboxy derivative of PEG
(compound XIX) (2 g, .apprxeq.1 mmol) and N-hydroxysuccinimide
(0.23 g, 2 mmol) were dissolved in methylene chloride-ethyl acetate
(4 mL, 1:1). The resulting solution was cooled in an ice-water bath
and treated with dicyclohexylcarbodiimide (DCC) (0.25 g, 1.2 mmol)
predissolved in ethyl acetate (1 mL). Within a few minutes the
solution became cloudy as dicyclohexylurea (DCU) appeared. After 2
hours the reaction mixture was filtered to remove DCU and
evaporated to dryness. The functionalized polymer was crystallized
from isopropanol and dried in vacuo over P.sub.2O.sub.5. Yield: 1.5
g (70%).
[0186] Titration of the product for active acyl content (Zaplipsky,
S., et al., POLYMERIC DRUGS (Dunn, R. L. and Ottenbrete, R. M.,
Eds.) American Chemical Society, pp. 91 (1991)) gave 4.810.sup.-5
mole/g (104% of the theoretical value).
[0187] The N-hydroxysuccinimide ester of
.alpha.-hydroxy-.omega.-carboxy-PEG (0.52 g, 0.2 mmol) was added to
a suspension of DSPE (0.14 g, 0.185 mmol) in chloroform (2 mL)
followed by addition of triethylamine (0.1 mL, 0.86 mmol). The
mixture was heated in a water bath at 55.degree. C. for 5 minutes,
during which time the solution became clear. TLC
(chloroform-methanol-water 90:18:2) on silica gel coated plates
showed complete conversion of DSPE into a new product, which gave
no color when treated with ninhydrin. The solution was treated with
an equivalent amount of acetic acid to neutralize the TEA and the
neutralized solution was evaporated to dryness. The residue was
dissolved in water and extensively dialyzed through a 300,000 MWCO
cellulose acetate membrane at 4.degree. C., filtered (pore size 0.2
.mu.m) and lyophilized, yielding pure compound XXIV (360 mg,
.apprxeq.70%).
[0188] This compound may then be further reacted with DSC to form a
PEG-derivatized DSPE lipid containing an .alpha.-succinimidyl
carbonate group.
EXAMPLE 5
Preparation of DSPE-PEG-Hydrazide (Compound XXXII)
[0189] A. Preparation of .omega.-Hydroxy Acid Derivative of PEG,
.alpha.-(Hydroxyethyl)-.omega.-(carboxymethyl-aminocarbonyl)oxy-poly(oxye-
thylene) (Compounds XIX and XXIX)
[0190] Polyethylene glycol (Fluka, PEG-2000, 42 g, 42 mequiv OH) is
dissolved in toluene (200 mL), azeotropically dried (Zalipsky, S.,
et al., Int. J. Peptide Res. 30:740 (1987)) and treated with ethyl
isocyanotoacetate (2.3 mL, 21 mmol) and triethylamine (1.5 mL, 10
mmol). The reaction mixture is stirred overnight at 25.degree. C.
and the solution is then evaporated to dryness. The residue is
dissolved in 0.2 M NaOH (100 mL) and any trace of toluene is
removed by evaporation. The solution is maintained at pH 12 with
periodical dropwise addition of 4 M NaOH.
[0191] When the solution pH is stabilized at pH 12, the solution is
acidified to pH 3.0 and the product is extracted with methylene
chloride (100 mL.times.2). TLC on silica gel (isopropyl
alcohol/H.sub.2O/conc. ammonia 10:2:1) gives a typical chromatogram
of partially carboxylated PEG (Zalipsky, et al., 1990) consisting
of unreacted PEG (R.sub.f=0.67), monocarboxylated derivative
(R.sub.f=0.55) and dicarboxylated derivative of the polymer
(R.sub.f=0.47). This solution is dried over anhydrous MgSO.sub.4,
filtered and evaporated to dryness. The PEG mixture is dissolved in
water (50 mL). One-third of this solution (30 mL.apprxeq.14 g of
derivatized PEG) is loaded onto DEAE-Sephadex A-25 (115 mL of gel
in borate form). After the underivatized PEG is washed off the
column with water (confirmed by negative poly(methacrylic acid),
PMA, test) (Zalipsky, et al., 1990), a gradient of ammonium
bicarbonate (2-20 mM at increments of 1-2 mM every 200 mL) is
applied, and 50 mL fractions are collected. Early eluting
fractions, e.g., fractions 1-25, typically contain only PEG
monoacid as determined by PMA and TLC analyses. These fractions are
then pooled, concentrated to .apprxeq.70 mL, acidified to pH 2 and
extracted with methylene chloride (50 mL.times.2). The
CH.sub.2Cl.sub.2 solution is dried over anhydrous MgSO.sub.4,
concentrated and poured into cold stirring ether. The precipitated
product (compound XXIX) is dried in vacuo. Yield: 7 g. Titration of
carboxyl groups gives 4.610.sup.-4 mequiv/g (97% of theoretical
value).
[0192] B. Preparation of Compound XXX
[0193] Compound XXIX (5 g, 2.38 mmol) and tert-butyl carbazate
(0.91 g, 6.9 mmol) are dissolved in CH.sub.2Cl.sub.2-ethyl acetate
(1:1, 7 mL). The solution is cooled on ice and treated with DCC
(0.6 g, 2.9 mmol) predissolved in the same solvent mixture. After
30 minutes the ice bath is removed and the reaction is allowed to
warm to room temperature and stirred for an additional 3 hours. The
reaction mixture is filtered to remove dicyclohexylurea and the
resulting filtrate is evaporated to produce a crude residue. The
residue is recovered and purified by two precipitations from ethyl
acetate-ether (1:1) and dried in vacuo over P.sub.2O.sub.5. Yield:
5.2 g, 98%. TLC of the product reveals one spot (R.sub.f=0.68) with
an R.sub.f value different from that of the starting material
(R.sub.f=0.55). H-NMR (CDCl.sub.3): .delta. 1.46 (s, t-Bu, 9H);
3.64 (s, PEG, 178H) 3.93 (br. d, J=4.5, CH.sub.2 of Gly, 2H); 4.24
(t, CH.sub.2--OCO-Gly, 2H) ppm. .sup.13C-NMR (CDCl.sub.3): .delta.
28.1 (t-Bu); 43.4 (CH.sub.2 of Gly); 61.6 (CH.sub.2OH); 64.3
(CH.sub.2OCONH); 69.3 (CH.sub.2CH.sub.2OCONH); 70.5 (PEG); 72.4
(CH.sub.2CH.sub.2OH); 81.0 (CMe.sub.3); 155.1 (C.dbd.O of Boc);
156.4 (C.dbd.O of Gly urethane; 168.7 (C.dbd.O of Gly hydrazide)
ppm.
[0194] C. Preparation of Compound XXXI
[0195] The .omega.-hydroxy Boc-hydrazide derivative of PEG
(compound XXX, 5 g, 2.26 mmol) is dissolved in pyridine (1.1 mL),
CH.sub.2Cl.sub.2 (5 mL) and CH.sub.3CN (2 mL) and treated with
disuccinimidyl carbonate, DSC (1.4 g, 5.5 mmol). The reaction
mixture is stirred at 25.degree. C. overnight. The mixture is then
filtered to remove solids and slowly added to cold ethyl ether (100
mL). The precipitated product is dissolved in warm ethyl acetate
(45 mL), chilled and mixed with equal volume of ethyl ether. The
precipitate is collected by filtration and dried in vacuo over
P.sub.2O.sub.5. Yield of compound XXXI: 4.8 g, 90%.
[0196] Succinimidyl carbonate group content 4.1510.sup.-4 mequiv/g
(98% of theoretical value) is determined by titration (Zalipsky, et
al., 1991). H-NMR (CDCl.sub.3): .delta. 1.46 (s, t-Bu, 9H); 2.83
(s, succinimide); 3.64 (s, PEG, 178H); 3.79 (t,
CH.sub.2CH.sub.2OCO.sub.2-Su); 3.93 (br. d, J=4.5, CH.sub.2 of Gly,
2H); 4.24 (t, CH.sub.2--OCO-Gly, 2H); 4.46 (t,
CH.sub.2OCO.sub.2-Su) ppm.
[0197] D. Preparation of Compound XXXII
[0198] To prepare the DSPE-PEG-hydrazide, a slight excess of
succinimidyl carbonate Boc-protected PEG-glycine hydrazide
(compound XXXI) is reacted with DSPE suspended in chloroform in the
presence of triethylamine. The lipid derivative is quickly (5-10
minutes) solubilized during progress of the reaction. Excess
heterobifunctional PEG is removed by dialysis using a 300,000 MWCO
cellulose ester dialysis membrane from Spectrum. The recovered
lipid conjugate is subjected to conventional Boc-deprotection
conditions (4M HCl in dioxane for 30 minutes) and then further
purified by recrystallization. H-NMR (CDCl.sub.3): .delta. 0.88 (t,
CH.sub.3, 6H); 1.59 (t, CH.sub.2CH.sub.2CO, 4H); 2.84 (t,
CH.sub.2CO, 4H); 3.64 (s, PEG, 180H); 4.0 (t); 4.2 (m,
CH.sub.2OCO--NH.sub.2); 4.4-4.3 (two doublets); 5.2 (g, CH of
glyceride).
EXAMPLE 6
Preparation of Liposomes with Covalently Bound
.beta.-Galactosidase
[0199] The maleimide of the DSPE carbamide of polyoxyethylene
bis(amine) (3500-DSPE) was prepared as in Example 1.
.beta.-Galactosidase was purchased from Pierce (Rockford, Ill.).
Enzyme assays with o-nitrophenyl galactose were performed
essentially by monitoring the development of the colored product
with an extinction coefficient of 4467 at 413 nanometers in 0.1 N
sodium hydroxide. The assay mixture consisted of 86 mM sodium
phosphate pH 7.3, 1 mM magnesium chloride, 50 mM
beta-mercaptoethanol and 2.3 mM o-nitrophenyl galactose and product
formation was monitored for 10 to 15 minutes in the linear range of
the assay.
[0200] Liposomes (MLV's) were prepared according to standard
methods with one of the compositions indicated in Table 4. The
liposomes were sized by extrusion through a polycarbonate membrane
to 200 nm. TABLE-US-00010 TABLE 4 "Phenotype" Mol % PEG- PEG- DSPE
Crosslinker .alpha.T Ch Pc Crosslinker DSPE PG - - 1 33 61 - - 5 +
- 1 33 61 - 5 - - + 1 33 56 5 - 5 + + 1 33 56 5 5 -
where .alpha.-T=.alpha.-tocopherol (antioxidant), Ch=cholesterol,
PC=partially hydrogenated egg PC (IV 40), crosslinker=the maleimide
derivative of PEG-3500-DSPE, and PG=egg phosphatidyl glycerol. In
addition, all liposome preparations were "spiked" with a
.sup.3H-DPPC tracer. The total lipid concentration in each
preparation, after hydration in PBS (50 mM sodium phosphate pH 7.2,
50 mM sodium chloride, was 2 mM.
[0201] Crosslinking reactions were performed by adding enzyme
solution to the liposomes (final protein concentration=0.5 mg/mL)
and incubating the suspension overnight at ambient temperature with
gentle shaking. Unreacted crosslinker was then quenched with 10 mM
2-mercaptoethanol (2-ME) for 30-60 minutes at 37.degree. C.
Liposomes were separated from unconjugated protein by flotation
through a metrizamide gradient: the sample was brought to 30% (w/v)
metrizamide and transferred to an SW60Ti tube, 20% metrizamide was
layered above, then PBS was added on top to provide an aqueous
interface. Gradients were centrifuged at 45,000 rpm for 60 minutes
at 4.degree. C., then each liposomal band, easily visible at the
PBS interface, was collected and transferred to dialysis tubing.
Dialysis proceeded overnight at 4.degree. C. against two changes of
PBS. Removal of the metrizamide was necessary because it inhibits
.beta.-galactosidase activity significantly even at 1% (w/v)
concentration.
EXAMPLE 7
Liposome Blood Lifetime Measurements of Hydrazide
End-functionalized PEG Liposomes
[0202] A. Preparation of Hydrazide End-functionalized Liposomes
[0203] Hydrazide PEG-DSPE composed of PEG, end-functionalized with
a hydrazide group, and distearyl-PE was prepared as described. The
hydrazide PEG-DSPE lipid was combined with partially hydrogenated
egg PC (PHEPC) and cholesterol in a lipid:lipid:lipid mole ratio of
about 0.15:1.85:1 and the lipid mixture was hydrated. Generally,
lipid hydration occurred in the presence of desferal mesylate,
followed by sizing to 0.1 micron, and removal of non-entrapped
desferal by gel filtration with subsequent loading of Ga-oxide into
the liposomes. The unencapsulated Ga was removed during passage
through a Sephadex G-50 gel exclusion column. Both compositions
contained 10 micromoles/mL in 0.15 M NaCl, 5 mM desferal.
[0204] A second lipid mixture was prepared in a similar manner but
with HSPC (hydrogenated serum phosphatidylcholine) instead of
PHEPC.
[0205] B. Measuring Blood Circulation Time and Tissue Levels
[0206] In vivo studies of liposomes were performed in laboratory
rats weighing 200-300 g each. These studies involved tail vein
injection of liposome samples at about 10-20 micromolar
phospholipid/kg body weight. Blood samples were obtained by
retroobital bleeding at defined times. The animals were sacrificed
after 24 hours and tissues removed for label quantitation. The
weight and percent of the injected dose in each tissue was
determined. The studies were carried out using .sup.67Ga-desferal
loaded liposomes and radioactivity was measured using a gamma
counter. The percent of the injected dose remaining in the blood at
several time points up to 24 hours, and in selected tissues at 24
hours was determined as follows.
[0207] 1. Plasma Kinetics of Hydrazide-PEG Liposomes.
[0208] The above-described liposome composition (0.4 mL) was
intravenously administered and at times 0, 0.25, 1, 3, or 5 and 24
hours after injection, blood samples were removed and assayed for
the amount of Ga-desferal present in the blood, expressed as a
percentage of the amount measured immediately after injection.
[0209] Hydrazide-PEG liposomes have a blood halflife of about 15
hours, and nearly 30% of the injected material was determined to be
present in the blood after 24 hours.
[0210] 2. 24 Hour Tissue Levels
[0211] Studies to determine the distribution of gallium-labelled
liposomes in selected tissues 24 hours after intravenous injection
were performed. The liposome composition (0.4 mL) was intravenously
administered as described in 7B above. The percent dose remaining
in tissues 24 hours after intravenous administration is shown in
Table 3.
[0212] While the invention has been described with reference to
specific methods and embodiments, it will be appreciated that
various modifications and changes may be made without departing
from the invention.
Sequence CWU 1
1
10 1 15 PRT Artificial Sequence synthetic peptide 1 Arg Ile Gln Arg
Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Lys 1 5 10 15 2 24 PRT
Artificial Sequence synthetic peptide 2 Asn Asn Thr Arg Lys Ser Ile
Arg Ile Gln Arg Gly Pro Gly Arg Ala 1 5 10 15 Phe Val Thr Ile Gly
Lys Ile Gly 20 3 8 PRT Artificial Sequence synthetic peptide 3 Arg
Ala Phe Val Thr Ile Gly Lys 1 5 4 13 PRT Artificial Sequence
synthetic peptide 4 Thr Lys Gly Pro Gly Arg Val Ile Tyr Ala Thr Gly
Gln 1 5 10 5 13 PRT Artificial Sequence synthetic peptide 5 His Ile
Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys Asn 1 5 10 6 5 PRT
Artificial Sequence synthetic peptide 6 Tyr Ile Gly Ser Arg 1 5 7 9
PRT Artificial Sequence synthetic peptide 7 Cys Asp Pro Gly Tyr Ile
Gly Ser Arg 1 5 8 5 PRT Artificial Sequence synthetic peptide 8 Gly
Arg Gly Asp Ser 1 5 9 10 PRT Artificial Sequence synthetic peptide
9 Arg Gly Asp Ser Gly Tyr Ile Gly Ser Arg 1 5 10 10 5 PRT
Artificial Sequence synthetic peptide 10 Tyr Cys Gly Ser Arg 1
5
* * * * *